Validation of miRNAs and Their Target Gene Expression by qRT-PCR

Total RNA was extracted from Control, H₂S, H₂S + AS, and AS treated roots according to the standard protocol of Trizol (Invitrogen, CA, United States). Total RNA was tailed addition reaction and first strand cDNA synthesis employed TransScript^® miRNA RT Enzyme Mix and 2 × TS miRNA Reaction Mix (TransGen Biotech, Beijing, China). The cDNA template for miRNA target gene qRT-PCR was synthesized using the TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (TransGen Biotech, Beijing, China). PerfectStart^TM Green qPCR Super Mix was used for qRT-PCR (TransGen Biotech, Beijing, China). Fifteen miRNAs and nine target genes were selected to conduct qRT-PCR and then verify the miRNA expression revealed by the RNA-seq. qRT-PCR was performed following the method used by Li et al. (2020). U6 and 18S rRNA were used as an internal standard to normalize the expression of miRNA and target genes. The roots from the Control treatment were taken as a reference sample and the relative expression level of genes was set to 1. All primers, including miRNAs and their targets in the qRT-PCR experiments, are shown in Supplementary Tables 3, 4.